Apparent calcium spark properties and fast-scanning 2D confocal imaging modalities.

Ca sparks are instrumental to understand physiological and pathological Ca signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120 Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the "true" Ca spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.